Bacillus anthracis is Gram-positive, 4-8 μm in length and 1-1.5 μm in width, making it the largest among pathogens, is square-ended, and sometimes forms long chains. B. anthracis is non-motile without flagella, and forms spores in unfavorable environments The spores can survive for 24 hours in air and even for 100 years in the soil, and have properties of high resistance to heat, sunlight, disinfecting agents, and the like.
Anthrax is a disease caused by a spore-forming bacterium belonging to the genus Bacillus, Bacillus anthracis. Since anthrax is actually rare in humans, studies involving anthrax have been not actively performed. Anthrax most commonly occurs in domestic animals (cattle, sheep, goats, camels, antelopes, and other herbivores), and often occurs in livestock workers who are exposed to infected livestock, or in people when they ingest products made from infected livestock. However, due to its high potential use for purposes of biological terrorism, B. anthracis has recently been classified by the American CDC (Centers for disease control and prevention) as a pathogenic microorganism of Category A, which has high potential for use in terrorism.
Anthrax infection may occur mainly in three forms: cutaneous (skin), inhalation (pulmonary), and intestinal. Among them, inhalation infection is most lethal. Initial symptoms of inhalation anthrax may resemble a common cold and include fever, difficulty in breathing, coughing, headaches, vomiting, chilling, abdominal pain, and chest discomfort After several days, the symptoms may progress to severe breathing problems and shock. Inhalation anthrax is usually fatal. About 20% of all cutaneous infection cases are fatal, and intestinal infection results in a 25-60% death rate. Inhalation infection is more frequently fatal.
In the case of inhalation anthrax, B. anthracis is drawn in its dormant spore state into the lungs through the respiratory tract, and is ingested by macrophages in the alveoli. The spores germinate within the macrophages, and are carried to lymph nodes, where they multiply. The bacterial cells then get into the bloodstream, and begin reproducing continuously and producing toxins, causing lethal symptoms (Maynard et al., Nature Biotechnology 2002 20:597-601).
B. anthracis produces anthrax toxin through a pXO1 plasmid. Anthrax toxin is composed of three distinct proteins: protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). The protective antigen, consisting of four folding domains, binds to the anthrax toxin receptor (ATR) on the cell surface through its domain 4. PA is then cleaved at the site of domain 1 by furin-like protease on the cell surface to produce PA63, releasing an N-terminal 20-kDa fragment The activated form of PA, PA63, oligomerizes into a heptamer, [PA63]7, to generate regions capable of binding to LF or EF. The PA63 heptamer combines with either LF or EF to form either lethal toxin (LeTx) or edema toxin (EdTx).
The PA63 heptamer-LF/EF complex is endocytosed into the cytoplasm, and fused with lysosome. The PA63 heptamer undergoes conformational changes at low pH, resulting in the release of LF and EF into the cytoplasm. In the cytoplasm, LF acts as a zinc-dependent metalloprotease which cleaves mitogen-activated protein kinase kinases. This cleavage disrupts the intracellular signal transduction pathway, resulting in the lysis of macrophages. EF is a calcium/calmodulin-dependent adenylate cyclase, which causes increased levels of intracellular cAMP levels, leading to swelling and local inflammation, which are generally not lethal.
At present, several antibiotics, such as penicillin, doxycycline, and fluroquinolones, are used for the treatment of anthrax infections. However, antibiotic treatment cannot be applied to antibiotic-resistant anthrax strains. In particular, this antibiotic treatment is not suitable for use in biochemical terrorism or biochemical warfare, which uses antibiotic-resistant strains. Also, since antibiotics cannot inhibit the action of anthrax toxin, anthrax is highly fatal if antibiotics are not administered at early stages of infection. Unfortunately, anthrax is difficult to diagnose and treat at early stages because it initially presents with cold-like symptoms.
Vaccines, whose major component is PA, have been developed and are currently used for preventing anthrax in the USA and Great Britain. However, since the vaccines have not been proven completely safe, their application is allowed only to army personnel and some persons who are highly liable to be exposed to B. anthracis. In addition, since a period of at least several months is required to acquire sufficient immunity, vaccines are actually impossible to apply in emergency situations such as in the event of biochemical terrorism. Thus, there is an urgent need for the development of therapeutic and preventive approaches, other than antibiotics, which can be applied to such situations.
Passive immunization using antibodies is a very effective strategy for toxin neutralization. In fact, the development of antibodies capable of neutralizing botulinum toxin and ricin in addition to anthrax toxin is in progress (Rainey et al., 2004 Nature reviews of Microbiology 2: 721-726). Several research groups revealed through studies using cells and small animals, such as guinea pigs, rats, mice, and hamsters, that antisera are very effective in neutralizing anthrax toxin.
Many attempts have recently been made to neutralize anthrax toxin using monoclonal antibodies against protective antigen (PA) and lethal factor (LF), and such attempts were reported to be successful in practice (Kobiler et al., 2002 Infection and Immunity 70:544-550; Cirino et al., 1999 Infection and Immunity 67:2957-2963; Beedham et al., 2001 Vaccine 19:4409-4416). Antibody-based neutralization of anthrax toxin may occur through a mechanism, such as binding inhibition between PA and its cellular receptor, inhibition of cleavage of PA, binding inhibition between PA and LF, and inhibition of the action of LF. For example, a monoclonal antibody, LF8, capable of neutralizing anthrax toxin, inhibits lethal toxin formation by binding to the PA binding domain of LF or near this domain (Zhao et al., 2003 Human Antibodies 12:129-135).
Taking the importance of antibodies into consideration, there is a need for the development of monoclonal antibodies having high specificity and affinity to antigens and thus being capable of effectively neutralizing anthrax toxin, leading to the effective prevention and treatment of anthrax.
In this regard, the present inventors selected hybridoma cells that secrete monoclonal antibodies capable of neutralizing anthrax toxin by binding to the lethal factor, and found through in vitro cell and in vivo animal studies using Fisher rats that the antibodies produced by the hybridomas have strong toxin-neutralizing activity. The present inventors also found that the antibodies have high affinity to their antigen and exhibit a preventive effect before exposure to anthrax toxin as well as a therapeutic effect after exposure to anthrax toxin. The present inventors further found that the antibodies have a neutralization mechanism different from that of a conventional antibody, LF8, and have higher cytotoxicity-neutralizing activity than the LF8 antibody. The present inventors also identified amino acid sequences of heavy chain and light chain variable regions of the antibodies and CDR regions of the variable regions, thereby leading to the present invention.